Voluntary exercise preserves visual function and reduces inflammatory response in an adult mouse model of autosomal dominant retinitis pigmentosa

Whole-body physical exercise has been shown to promote retinal structure and function preservation in animal models of retinal degeneration. It is currently unknown how exercise modulates retinal inflammatory responses. In this study, we investigated cytokine alterations associated with retinal neuroprotection induced by voluntary running wheel exercise in a retinal degeneration mouse model of class B1 autosomal dominant retinitis pigmentosa, I307N Rho. I307N Rho mice undergo rod photoreceptor degeneration when exposed to bright light (induced). Our data show, active induced mice exhibited significant preservation of retinal and visual function compared to inactive induced mice after 4 weeks of exercise. Retinal cytokine expression revealed significant reductions of proinflammatory chemokines, keratinocyte-derived chemokine (KC) and interferon gamma inducible protein-10 (IP-10) expression in active groups compared to inactive groups. Through immunofluorescence, we found KC and IP-10 labeling localized to retinal vasculature marker, collagen IV. These data show that whole-body exercise lowers specific retinal cytokine expression associated with retinal vasculature. Future studies should determine whether suppression of inflammatory responses is requisite for exercise-induced retinal protection.


Induction of the I307N Rho degeneration
Animals were dark adapted overnight and atropine eye drops (0.2% diluted from 1% atropine ophthalmic solution, Akorn Inc., Lake Forest, IL; diluted with Refresh Tears (Allergan, Irvine, TX)) were administered twice, with the last eye drops applied 30 min before toxic light exposure.To induce I307N Rho degeneration, animals were individually housed in shoebox containers with a white light emitting diode (LED) light panel (LED500A; Fancierstudio, Hayward, CA) fitted to a standard mouse cage as previously described 19 .Mice were exposed to either bright light (6000 lx, induced) to induce retinal degeneration or dim light (50 lx, uninduced) for 5 min.Room and light box temperatures were closely monitored to ensure animal welfare.Light induction occurred between 10 and 11 AM.Following light induction, mice were returned to their home cages under normal lighting conditions for the remainder of the experiment.

Electroretinography (ERG)
Retinal function (n = 11-13 per group) was measured with a commercial electroretinography (ERG) system (Bigshot; LKC Technologies, Gaithersburg, MD) as previously described by our group 16,19 .After overnight dark adaptation, mice were anesthetized (ketamine [80 mg/kg]/xylazine [16 mg/kg]).All procedures were performed under dim red light.The pupils were dilated (1% tropicamide; Alcon Laboratories, Ft. Worth, TX) and corneas were anesthetized (1% tetracaine; Alcon Laboratories, Ft. Worth, TX).Body temperature was maintained with a heating pad (ATC 1000; World Precision Instruments, Sarasota, FL) for the duration of the session.The ERG protocol consisted of a ten-step series of full-field flash stimuli produced by a Ganzfeld dome under both darkadapted scotopic conditions (− 3.0 to 2.1 log cd s/m 2 ) to test rod and rod/cone pathways and light-adapted photopic conditions to test cone pathway function.(2.0 log cd s/m 2 presented as 6.1 Hz flicker with a 30 cd/m 2 background light).Custom gold-loop wire electrodes were placed on the center of each eye through a layer of 1% methylcellulose to measure the electrical response of the eye to each flash.Both reference and ground platinum needle reference electrodes (1 cm; Natus Medical Incorporated, Pleasanton, CA) were inserted subcutaneously in each cheek and the tail, respectively.Once ERG was completed, mice were given an IP injection of atipamezole (1 mg/kg; Antisedan, Zoetis, Parsippany, NJ) to counteract the effects of xylazine 32 , administered saline eye drops and allowed to recover on a heating pad (37 °C) before being returned to housing.ERG responses from both eyes were averaged together.

Optomotor response (OMR)
A virtual OMR tracking system was used to measure visual function in all experimental groups (n = 11-16 per group; OMR; OptoMotry®; Cerebral Mechanics Inc., Lethbridge, AB, Canada) as previously described by our group and others [33][34][35][36] .Mice were placed on a circular platform in the center of a virtual reality chamber composed of four computer monitors, which present vertical sine wave gratings revolving at a speed of 12 deg/s.A video camera monitored the animals' behavior in real time throughout the experiment.The presence or absence of reflexive movements by the mouse's head were noted as it tracked the rotating gratings moving either in a clockwise or counter-clockwise direction.When mice became distracted, their attention was regained by gently tapping the instrument.OptoMotry® uses a staircase paradigm to automatically calculate spatial frequency and contrast sensitivity thresholds.To evaluate spatial frequency, contrast was held constant at 100% while spatial frequency gratings started with a frequency of 0.042 cyc/deg and adjusted over time with the presence or absence of head movements.To assess contrast sensitivity, spatial frequency was held constant at the peak of the contrast sensitivity curve at 0.064 cycles per deg (c/d), while contrast began at 100% and adjusted over time.The contrast sensitivity reported here was calculated as a reciprocal of the Michelson contrast from the screen's luminance (max + min/max − min), as previously described 37 .The OMR was assessed by a masked observer.The spatial frequency and contrast sensitivity thresholds were determined after three positive responses and calculated as the average of the reported values for both eyes and were compared across time points.

Histology and morphometrics of ocular sections
For histological analyses, eyes were enucleated and then immersion-fixed in 4% paraformaldehyde for 2 h at room temperature and rinsed with 0.1 M phosphate buffer.Posterior eyecups were processed through a graded alcohol series and embedded in plastic resin (Embed 812/DER 736, Electron Microscopy Science).Using an ultramicrotome (Reichert Ultracut, Leica, Inc., Buffalo Grove, IL) with a histo-diamond knife, sections (0.5 μm) were cut through the superior to inferior retina bisecting the optic nerve.Plastic embedded retinal sections were stained with 1% aqueous toluidine blue (Millipore Sigma, Burlington, MA) and imaged using a Leica DMLB microscope with a 40 × N Plan brightfield objective (Leica, Inc., Buffalo Grove, IL).Retinal spidergrams were constructed by plotting the number of photoreceptor nuclei present in the outer nuclear layer (ONL) and cone photoreceptor quantifications as a function of position in the retina relative to the optic nerve.ONL and cone nuclei were manually counted using Adobe Photoshop (24.0.0 release) within 100-μm-wide segments spaced at 250, 500, 750, 1000 and 1250 μm from the optic nerve head in the superior and inferior directions within whole retinal sections (n = 8 per group, 3 retinal sections quantified per animal).

Immunofluorescence
Eyes were fixed in methanol acetic acid, dehydrated, embedded in paraffin, and sectioned through the sagittal plane on a microtome (5 μm).Sections containing the optic nerve were selected for staining to ensure that consistent regions were examined between animals.The slides were deparaffinized across five Coplin jars with 100 mL of xylene for 2 min each, consecutively.Then the slides were rehydrated in a series of 100 mL ethanol solutions for 2 min each: 100%, 90%, 80%, 70%, 60%, and 50%.Following rehydration, retinal sections were blocked for 30 min at room temperature in 5% normal donkey serum in PBS with 0.01% sodium azide and 0.3% Triton X-100.Primary antibodies were diluted in 5% normal donkey serum in PBS with 0.01% sodium azide and incubations were performed overnight at 4 °C using KC monoclonal antibody (48415, Invitrogen, Waltham, MA, 1:100 dilution), IP-10 monoclonal antibody (sc-374092, Santa Cruz Biotechnology, Dallas, TX, 1:100 dilution), Gt X Collagen Type IV antibody (AB769, Millipore Sigma, Burlington, MA, 1:100 dilution) and cone arrestin (15282, Millipore Sigma, Burlington, MA, 1:100 dilution).Secondary antibodies were diluted in PBS and incubations were performed for 1 h at room temperature in the dark (Alexa Fluor® 647 donkey antimouse; 1:500, A32787, ThermoFisher Scientific, Waltham, MA; Alex Fluor® 568 donkey anti-goat; 1:500, A11057, ThermoFisher Scientific, Waltham, MA).Retinal sections were mounted with ProLong®Gold Antifade Reagent with DAPI (#8961, Cell Signaling Technology Inc., Danvers, MA).Retinal sections were imaged using a Nikon A1R HD25 confocal microscope with a Plano Apo 20 × NA 0.75 objective and compiled and quantified using ImageJ software.KC and IP-10 labeling quantifications are the result of positive KC and IP-10 labeling within the superior, inner and deep vascular retinal plexi (n = 6 animals per group, each symbol in the plots represents the average of three retinal sections per animal).Cone arrestin labeling quantification is the result of positive cone arrestin labeling within the outer/inner segments and outer nuclear layer (n = 8 per group, 3 retinal sections quantified per animal).

Multiplexed cytokine immunoassay
Whole retinal extract (excluding RPE, n = 4-9 animals per group) was flash frozen and lysed using a Bio-Plex cell lysis kit (Bio-Rad, Fort Worth, TX) according to manufacturer's protocol, with the addition of one cOmplete mini (Roche, San Jose, CA) protease inhibitor per 5 mL of buffer.Lysates were placed on an end-over-end rotator for 20 min and centrifuged at 13.2kRPM.Protein concentrations were determined using a Pierce BCA Protein Assay (Thermo Fisher, Waltham, MA).Samples were normalized to a concentration of 0.75 μg in 37.5 μL of Milliplex® MAP Assay Buffer (EMD Millipore, Burlington, MA) because this loading fell within a linear range of detectable analytes 38  www.nature.com/scientificreports/

Masking and statistical analysis
Sample size was determined based on our previously reported data 16,17,19 .The observers who analyzed the data were blinded to the experimental procedures and were masked to the specific treatment group from which sampling arose.This included semi-automated marking of ERG a-and b-waves (Matlab 2021b, Mathworks, Natick, MA), image acquisition, semi-automated quantification of KC and IP-10 fluorescence labeling in retinal sagittal sections (ImageJ software).Statistical analyses were performed using Graphpad Prism 9.0.0 (San Diego, CA).Two-way ANOVAs dividing groups by exercise and light exposure to test the interactive effects of the independent variables and Tukey's multiple comparison tests were performed on all visual and retinal functional data, ONL counts, cone photoreceptor counts, and fluorescence quantification and are presented as mean ± standard error of the mean (SEM).A Pearson correlation coefficient was computed to assess the linear relationship between individual animals KC and IP-10 expression, retinal function (scotopic a-and b-wave function and photopic b-wave function) and visual function (spatial frequency and contrast sensitivity).Multiplex cytokine data were analyzed using both univariate and multivariate techniques.For univariate analysis, active vs inactive samples were compared using an unpaired t-test with Welch's correction to account for differing sample sizes among the groups.To account for co-variance among cytokines, we also conducted a partial least squares discriminant analysis (PLSDA) using the ropls package in R (Bioconductor 39,40 ).To ensure the results of the PLSDA were robust to a subset of the samples (mice), we conducted a leave-n-out cross validation computing 100 iterations of PLSDA while each time leaving out four random samples (~ 15% of samples) then computed the standard deviation of loadings across all iterations.
All p-values lower than 0.05 were considered statistically significant.The ROUT method (with Q set to 1%) was used to detect outliers, of which none were detected.

Voluntary exercise conserved photoreceptor nuclei in active mice undergoing retinal degeneration
Outer nuclear layer (ONL) and cone photoreceptor quantifications were performed in retinal sagittal sections from all experimental groups.Consistent with our results measuring retinal and visual function, morphologic differences between active and inactive induced groups were apparent (Fig. 3a,c).Although photoreceptor loss was evident in both induced groups, active induced mice had significant preservation of photoreceptor nuclei compared to inactive induced mice (Fig. 3b; two-way ANOVA, F (3, 864) = 771.9,p < 0.0001, n = 8 per group, 3 retinal sections quantified per animal) and cone photoreceptors (Fig. 3d; two-way ANOVA, F (3, 864) = 300.3,p < 0.0001, n = 8 per group, 3 retinal sections quantified per animal).

Discussion
The protective effects of exercise for the prevention or treatment of neurodegenerative diseases are now welldocumented in human and animal studies 11,[13][14][15] .In this study, we tested whether an exercise regimen that protected against inherited retinal degeneration also altered retinal inflammatory responses.Determining how whole-body exercise alters the retinal cytokine network and inflammation in models of retinal degenerative diseases provides insight on how exercise-mediated interventions may halt and or slow vision loss.The eye, similar to the brain, is an immune privileged tissue.The retina reacts with a well-orchestrated and regulated multifaceted immune reaction in response to stress and insult 41 .Retinal degenerative diseases compromise the integrity of ocular immune privilege 42 .This results in overwhelming and non-resolving inflammation, ultimately moderating and driving disease progression.
In inherited retinal diseases, inflammatory responses have been generally considered as a secondary phenomenon associated with rod photoreceptor cell death, with the primary insult being induced by the causal genes (i.e., visual cycle protein mistrafficking, compromised photoreceptor structure integrity, inadequate RNA processing) 4,25 .However, the most effective neuroprotective treatments for adRP will also need to reduce this immune response to maintain maximal visual function.Inflammatory cytokines and chemokines elevated in RP are related to innate and acquired immunity 23,24 .In addition to maintaining retinal integrity, in this study we found both uninduced and induced active mice had significantly attenuated expression of cytokines KC and IP-10 and that their immunolabeling localized to retinal vasculature.
It is well-established that KC is a chemoattractant that recruits polymorphonuclear neutrophils to inflamed sites.In a study investigating the effects of treadmill exercise on cutaneous wound healing in aged mice, a significant reduction in KC expression was reported in dorsum tissue from exercised mice compared to inactive   mice 43 .This study suggested that exercise not only accelerated the wound healing process, but also induced an anti-inflammatory response in the wound 43 .To date, retinal Müller glia, microglia, RPE and vascular endothelial cells have all been reported to have KC immunoreactivity 44,45 .Additionally, activation of CD40 expressed in retinal endothelial cells and Müller glia has been shown to promote KC production 45 .Recently, an in vitro study investigating the immunological reaction of murine microglia cells incubated with lipofuscin isolated from human RPE reported elevated KC levels in the cell culture supernatant 46 .IP-10 is a pro-inflammatory cytokine that modulates angiogenesis and has been reported as a biomarker for age-related macular degeneration (AMD) [47][48][49] , another retinal disease characterized by photoreceptor degeneration.IP-10 was shown to be significantly elevated in all serum samples collected from subjects with different stages of both wet and dry AMD 48 .IP-10 labeling was abundant in the connective tissue matrix associated with choroidal neovascularization as well as neovascular endothelial cells in postmortem retinal tissues from AMD subjects 48 .Elevated expression of IP-10 and vascular growth factors VEGF and platelet derived growth factor (PDGF) have also been reported in the aqueous humor of AMD patients 50 .
In our study, we report diminished KC and IP-10 expression in retinal extract from active mice, both uninduced and induced compared to inactive groups.Furthermore, correlation coefficients revealed a moderate negative correlation between IP-10 expression and visual function outcome measurements.In retinal sections, KC and IP-10 were both localized to retinal vasculature through co-labeling with collagen IV.In future studies, analysis of retinal tissue at acute timepoints (12 h and 1, 3, and 5 days after induction) may provide further insight of the initial alterations exercise elicits on the retinal cytokine network during retinal degeneration in the I307N Rho mouse model.These studies could also explore the retinal and immune cell-types mediating exercise-induced Although photoreceptor loss was evident in both induced groups, active induced mice had consistently greater numbers of outer nuclear layers when compared to inactive induced groups.Retinal layers are as follows: outer segment (OS), inner segment (IS), outer nuclear layer (ONL), inner nuclear layer (INL) and ganglion cell layer (GCL).N = 8 per group, 3 retinal sections quantified per animal, ****p < 0.0001.Values are mean ± SEM. cytokine response.Incorporating animal knockout models or antagonists of the relevant cytokines or upstream phospho-protein signaling pathways may also identify targeted mechanisms that can be rapidly translated based on currently available small molecules and antibody therapies 51,52 .Additionally, investigating other animal models of retinal disease, such as diabetic retinopathy and glaucoma, would provide insight on similarities and/or differences in exercise-induced retinal cytokine responses.Future studies could also investigate how exercise may modify retinal epigenetic mechanisms, altering retinal transcriptional patterns, and potentially altering causal genes 53,54 .
We demonstrated that voluntary running wheel exercise provided significant preservation of retinal and visual function as well as conservation of photoreceptor nuclei number in an inducible model of adRP, I307N Rho.Our study provides the first evidence that exercise significantly attenuates retinal KC and IP-10 protein levels in both healthy and degenerating retinas.We further determined that KC and IP-10 labeling localized to retinal vasculature in all experimental groups.Correlation analyses revealed IP-10 expression had a significant moderately negative association with all retinal and visual functional assays, whereas the relationship between KC expression and retinal functional measurements was not as robust.This study demonstrates that whole-body exercise can alter the retinal cytokine response in both healthy and degenerating retinas.These data provide evidence that supports exercise as a potential effective therapeutic strategy for inherited retinal degenerations, which currently have minimal treatment options.

Figure 1 .
Figure 1.Voluntary running wheel exercise preserved retinal function in induced I307N Rho mice.To test the effects of voluntary exercise on retinal and visual function, we performed electroretinography (ERGs) and optomotor response (OMRs) measurements at baseline, 2-and 4-weeks post light induction (a).To ensure exercise-induced retinal neuroprotection was achieved as demonstrated in our labs previously, electroretinography (ERG) recordings were performed to non-invasively measure retinal function.Representative ERG waveforms are shown from dark-adapted stimuli (2.1 log cd s/m 2 ; b).Active induced mice had significant preservation of a-wave (c), b-wave (d) and photopic b-wave amplitudes (e) compared to inactive induced mice.a-and b-waves show rod photoreceptor and inner retinal function, respectively at 2-weeks post light induction.The photopic ERG measures cone photoreceptor function.Two-way ANOVA with Tukey's multiple comparison analysis was performed.N = 11-13 per group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Values are mean ± SEM.IF immunofluorescence.

Figure 3 .
Figure 3. Active induced I307N Rho mice maintained photoreceptor nuclei.Retinal sections from all experimental groups (a,c) were used to quantify photoreceptor nuclei present in the outer nuclear layer (ONL).Morphometric analyses were constructed by plotting the quantification of ONL nuclei and cone photoreceptors by cone arrestin labeling as a function of position in the retina relative to the optic nerve (μm) spidergram (b,d).Although photoreceptor loss was evident in both induced groups, active induced mice had consistently greater numbers of outer nuclear layers when compared to inactive induced groups.Retinal layers are as follows: outer segment (OS), inner segment (IS), outer nuclear layer (ONL), inner nuclear layer (INL) and ganglion cell layer (GCL).N = 8 per group, 3 retinal sections quantified per animal, ****p < 0.0001.Values are mean ± SEM.

Figure 4 .
Figure 4. Cytokine expression elevated in inactive vs active mice.(a) Measured cytokines (columns, z-scored) from retinal extracts of all experimental animals (rows).(b) Partial least squares discriminant analysis (PLSDA) identified a profile of cytokines, LV1, correlated with active cases (negative) or inactive cases (positive).(c,d) LV1 separated inactive induced and uninduced cases to the right (unfilled shapes) and active induced and uninduced cases to the left (filled shapes).(e) Univariate analysis revealed significantly higher expression of KC, IP-10, and IL-13 in inactive vs active mice (*p < 0.05, unpaired t-test with Welch's correction) and trending higher expression in inactive mice for VEGF, G-CSF, and MIG.N = 4-9 per group, each symbol in the plots represent retina from individual animals.

Figure 5 .
Figure 5. KC and IP-10 labeling localized to retinal vasculature.To determine the retinal cell type(s) expressing KC and IP-10, immunofluorescence on sagittal retinal sections from experimental groups was performed.Immunofluorescence revealed KC (a-d, green) and IP-10 (f-i, green) labeling localized to retinal vasculature as shown with co-labeling for collagen IV (a-i, red).We also observed inactive induced mice had a significant increase in both KC (e, p = 0.0110) and IP-10 (j, p = 0.0220) compared to uninduced mice.N = 6 animals per group, each symbol in the plots represents the average of three retinal sections per animal.KC and IP-10 labeling quantification are the result of positive KC and IP-10 labeling within the superior, inner and deep vascular plexi, *p < 0.05.Values are mean ± SEM.